Derivatisation of erythropoietin (EPO)

ABSTRACT

The present invention relates to a compound which is a polysaccharide derivative of EPO, or of an EPO like protein, wherein the polysaccharide is anionic and comprises between 2 and 200 saccharide units. The present invention also relates to pharmaceutical compositions comprising the novel compounds, and methods for making the novel compounds.

The present invention is a divisional of U.S. patent application Ser.No. 13/646,605 filed on Oct. 5, 2012, which is a continuation of U.S.patent application Ser. No. 12/375,008 filed on Jan. 23, 2009, nowissued as U.S. Pat. No. 8,299,026, which is the national stage entry ofPCT/GB2007/002841, filed on Jul. 25, 2007, which claims priority to EP06117830.7, filed on Jul. 25, 2006, all of which are hereby incorporatedby reference.

The present invention relates to novel polysaccharide derivatives of EPOand methods for producing such derivatives. The derivatives are usefulfor improving the stability, pharmacokinetics and pharmacodynamics ofEPO.

Erythropoietin is a glycoprotein hormone and is a cytokine forerythrocyte (red blood cell) precursors in the bone marrow. Also calledhematopoietin or hemopoietin, it is produced by the kidney, andregulates red blood cell production.

Erythropoietin is the main regulator of erythropoiesis in the body.(Martindale, 1996). Recombinant human erythropoietin is commerciallyavailable in forms known as epoetin alfa and epoetin beta. These areused in the management of anaemia in patients with chronic renalfailure. Epoetin gamma is also available. All have the same 165 aminoacid sequence, but differ in their glycosylation pattern.

Recombinant human erythropoietin should be used with caution in patientswith hypertension, a history of seizures, thrombocytosis, chronic liverfailure, ischaemic vascular disease, or in patients with malignanttumors. Epoetin alfa and beta exhibit some differences in theirpharmacokinetics, possibly due to differences in glycosylation and inthe formulation of the commercial preparations. Epoetin alfa is slowlyand incompletely absorbed following subcutaneous injection and abioavailability of about 10 to 50% relative to intravenousadministration has been reported. Epoetin beta is also slowly andincompletely absorbed and its absolute bioavailability has been reportedto be around 40%.

Attempts have been made to derivatise EPO to improve its pharmacokineticproperties. There is a product under development by Roche, known as CERA(Constant Erythropoiesis Receptor Activator), which is apolyethyleneglycol derivatised form of EPO. This has been shown to havea longer half-life than EPO, reducing the necessity of frequentinjections. A further novel erythropoiesis stimulating agent isHematide, a novel, PEGylated, synthetic peptide for the treatment ofanaemia associated with chronic kidney disease and cancer. This isdescribed further by Fan et al (2006).

Other forms of EPO have also been developed, such as Darbepoetin, ahyperglycosylated analogue of recombinant human erythropoietin which hasaround a three-fold longer terminal half life after i.v. administrationthan recombinant human EPO and the native hormone.

EP 1219636 describes modified muteins of EPO produced from amicroorganism with a prolonged plasma half-life in the circulation. Acell-free protein synthesis technique is used to produce a mutein of EPOwith an unnatural amino acid which may be reacted with a modifier suchas PEG or a polysaccharide. Generally, PEG is attached to a freesulfhydryl group in the muteins of EPO.

U.S. Pat. No. 7,128,913 is directed to N-terminal conjugates of EPO withPEG. The conjugates have an increased circulating half life and plasmaresidence time.

US 2004/0082765 describes an improved method for generatingPEG-conjugated EPO. The inventors found that a composition of conjugateshaving 1-3 linear PEG molecules per rhEPO molecule provided the mostsustained efficacy.

U.S. Pat. No. 7,074,755 also addresses the problem of providing improvedbiologically active EPO conjugate compositions. The EPO is covalentlyconjugated to a non-antigenic hydrophilic polymer covalently linked toan organic molecule that increases the circulating serum half-life ofthe composition. The water-soluble polymer may be a polyalkylene oxide,a polyamide, or a carbohydrate, amongst others.

However, there has been no published work to date describing thederivatisation of EPO with anionic polysaccharides such as polysialicacid (PSA).

Polysialic acids (PSAs) are naturally occurring unbranched polymers ofsialic acid produced by certain bacterial strains and in mammals incertain cells. They can be produced in various degrees of polymerisationfrom n=about 80 or more sialic acid residues down to n=2 by limited acidhydrolysis or by digestion with neuraminidases, or by fractionation ofthe natural, bacterially derived forms of the polymer.

In recent years, the biological properties of polysialic acids,particularly those of the alpha-2,8 linked homopolymeric polysialicacid, have been exploited to modify the pharmacokinetic properties ofprotein and low molecular weight drug molecules. Polysialic acidderivatisation gives rise to dramatic improvements in circulatinghalf-life for a number of therapeutic proteins including catalase andasparaginase, and also allows such proteins to be used in the face ofpre-existing antibodies raised as an undesirable (and sometimesinevitable) consequence of prior exposure to the therapeutic protein.The alpha-2,8 linked polysialic acid offers an attractive alternative toPEG, being an immunologically invisible biodegradable polymer which isnaturally part of the human body, and which degrades, via tissueneuraminidases, to sialic acid, a non-toxic saccharide.

We have previously described methods for the attachment ofpolysaccharides (in particular PSA) to therapeutic agents such asproteins [U.S. Pat. No. 5,846,951; WO-A-0187922]. Some of these methodsdepend upon chemical derivatisation of the ‘non-reducing’ end of thepolymer to create a protein-reactive aldehyde moiety which reacts atprimary amine groups. A non-reducing sialic acid terminal unit, since itcontains vicinal diols, can be readily (and selectively) oxidised withperiodate to yield a mono-aldehyde form, which is much more reactivetowards proteins, and which comprises a suitably reactive element forthe attachment of proteins via reductive amination and otherchemistries. The reaction is illustrated in FIGS. 1 and 2 wherein;

FIG. 1 shows the oxidation of colominic acid (alpha-2,8 linkedpolysialic acid from E. coli) with sodium periodate to form aprotein-reactive aldehyde at the non-reducing end; and

FIG. 2 shows the selective reduction of the Schiff's base with sodiumcyanoborohydride to form a stable irreversible covalent bond with theprotein amino group.

Unintentional by-products may be generated during the conventionalconjugation reactions described above by reaction of the colominic acidwith side chains of amino acids, for instance. These may be sufficientto be troublesome in the manufacture of chemically defined conjugatesrequired by regulatory authorities for therapeutic use in man andanimals.

It is not straightforward to purify the intended reaction product (forinstance the monopolysialylated product) away from the variousunintended products, since the physicochemical characteristics of mostof the reaction products are similar. This means that techniques such asion-exchange chromatography and gel-permeation chromatography (whichseparate on the basis of charge and size respectively) produce poorpurification profiles. This problem can be overcome by reducing theproduct complexity in the conjugation reaction. We have developed a newmethod for conjugation of polysaccharides to proteins whereby the highreactivity of the N-terminal of the protein can be utilised and whichavoids the product complexity obtained using the established method(FIGS. 1 and 2) of reductive amination of proteins with periodateoxidised natural colominic acid.

In view of the prior art, there is a need to provide improvedderivatives of EPO which can be used in human and animal therapy andhave optimised stability, half lives and low toxicity. We have foundthat attaching polysaccharides such as PSAs to EPO imparts suchproperties and have thereby arrived at this invention. This is the firsttime that EPO linked to anionic polysaccharides has been described.

In accordance with a first aspect of this invention we provide acompound which is a polysaccharide derivative of EPO, or of an EPO likeprotein, wherein the polysaccharide is anionic and comprises between 2and 200 saccharide units.

Hereinafter, when using the term EPO, we also intend to cover EPO-likeproteins. By EPO-like protein, we mean a protein which has an activityequivalent to that of EPO. EPO regulates erythrocyte production, asdetailed above. The activity of EPO or an EPO-like protein can bemeasured using a standard assay as described in Krystal (1983). Theactivity of EPO samples in inducing proliferation in vitro oferythrocyte progenitor cells isolated from the spleen of a mouse ismeasured. The mice have previously been rendered anaemic artificiallythrough I.P. injection of phenylhydrazine. In the assay, EPO is added toerythrocyte progenitors and the rate of DNA replication is measured bydetermining the rate of incorporation of ³H-thymidine. A protein isclassified as “EPO-like” if it induces 10-200% of the rate ofreplication compared to standard EPO from NIBSC. Typically, an EPO-likeprotein has at least 35% of the activity of standard EPO, andpreferably, at least 50% of the activity of standard EPO.

Mutants of EPO which have the requisite activity, as detailed above, mayalso be used. An “EPO-like” protein may also be referred to as an“EPO-homologue”. Whether two sequences are homologous is routinelycalculated using a percentage similarity or identity, terms that arewell known in the art. Sequences should be compared to the sequence ofthe human EPO precursor with swissprot accession number PO1588. Theactive EPO is residues 28-193 of this sequence. EPO homologue sequencesmay either be compared to the whole sequence of the human EPO precursorwith swissprot accession number PO1588, or residues 28-193 thereof.Preferably, EPO homologue sequences are compared to the active EPO, i.e.residues 28-193.

In this invention, homologues have 50% or greater similarity or identityat the nucleic acid or amino acid level, preferably 60%, 70%, 80% orgreater, more preferably 90% or greater, such as 95% or 99% identity orsimilarity at the amino acid level. A number of programs are availableto calculate similarity or identity; preferred programs are the BLASTn,BLASTp and BLASTx programs, run with default parameters (available onthe NCBI-NIH database). For example, 2 amino acid sequences may becompared using the BLASTn program with default parameters (score=100,word length=11, expectation value=11, low complexity filtering=on). Theabove levels of homology may be calculated using these defaultparameters.

The EPO may be glycosylated or non-glycosylated. When the EPO isglycosylated, the compound typically comprises 2-100 saccharide units.More typically, the compound comprises 10-80 saccharide units,preferably 20-60 saccharide units, most preferably 40-50 saccharideunits.

When the EPO is non-glycosylated, the compound typically comprises80-180 saccharide units, preferably 100-150 saccharide units, morepreferably 120-145, most preferably 130-140 units.

Preferably, the anionic polysaccharide has at least 2, more preferablyat least 5, most preferably at least 10, for instance at least 50saccharide units.

The anionic polysaccharide is preferably selected from polysialic acid,heparin, hyaluronic acid and chondroitin sulphate. Preferably, thepolysaccharide is polysialic acid and consists substantially only ofsialic acid units. However, the polysaccharide may have units other thansialic acid in the molecule. For instance, sialic acid units mayalternate with other saccharide units. Preferably, however, thepolysaccharide consists substantially of units of sialic acid.

Preferably, the compound is an N-terminal derivative of EPO or of anEPO-like protein, that is, the polysaccharide is associated with the EPOat its N-terminus. In this specification, by derivatisation at theN-terminus, we mean derivatisation at the N-terminal amine group of theEPO. Alternatively, however, the polysaccharide may be associated withthe EPO or EPO-like protein at a mid-chain amino acid, such as at theside chain of a lysine, cysteine, aspartic acid, arginine, glutamine,tyrosine, glutamic acid or histidine. Typically, the side chain is of alysine of cysteine amino acid.

Preferably the polysaccharide has a terminal sialic acid group, and asdetailed above, is more preferably a polysialic acid, that is apolysaccharide comprising at least 2 sialic acid units joined to oneanother through α-2-8 or α-2-9 linkages. A suitable polysialic acid hasa weight average molecular weight in the range 2 to 50 kDa, preferablyin the range 5 to 50 kDa. Most preferably, the polysialic acid isderived from a bacterial source, for instance polysaccharide B of E.coli KI, N. meningitidis, Maraxella liquefaciens or Pasteurellaaeruginosa or K92 polysaccharide from E. coli K92 strain. It is mostpreferably colominic acid from E. coli K1.

The polysialic acid may be in the form of a salt or the free acid. Itmay be in a hydrolysed form, such that the molecular weight has beenreduced following recovery from a bacterial source.

The polysaccharide, which is preferably polysialic acid may be materialhaving a wide spread of molecular weights such as having apolydispersity of more than 1.3, for instance as much as 2 or more.Preferably the polydispersity (p.d.) of molecular weight is less than1.3, more preferably less than 1.2, for instance less than 1.1. The p.d.may be as low as 1.01.

The EPO may be derivatised with more than one anionic polysaccharide.For instance, the EPO may be derivatised at both its N-terminus and atan internal amino acid side chain. The side chains of lysine, cysteine,aspartic acid, arginine, glutamine, tyrosine, glutamic acid, serine andhistidine, for instance, may be derivatised by an anionicpolysaccharide. The EPO may also be derivatised on a glycon unit.However, in a preferred embodiment of this invention, the EPO isderivatised at its N-terminus only.

In this specification, by derivatisation at the N-terminus, we meanderivatisation at the N-terminal amine group of the EPO.

The compound according to the first aspect of this invention may be acovalently-linked conjugate between the EPO and an anionicpolysaccharide. Other means of association between the polysaccharideand the EPO include electrostatic attraction. However, covalent bondingis preferred. The EPO may be covalently linked to the polysaccharide atits N-terminal amino acid. The covalent linkage may be an amide linkagebetween a carboxyl group and an amine group. Another linkage by whichthe EPO could be covalently bonded to the polysaccharide is via a Schiffbase. Suitable groups for conjugating to amines are described further inWO2006/016168.

In the invention the polysaccharide may be a naturally occurringpolysaccharide, or a derivative of a naturally occurring polysaccharide,for instance, a polysaccharide which has been derivatised by a reactionof one or more active groups on the saccharide residues, or which hasbeen covalently linked to a derivatising group at the end of thepolysaccharide chain.

The polysaccharide may be linked to the EPO via either its reducing ornon-reducing terminal unit. This means that one polysaccharide chain maybe linked to two EPO proteins, i.e. be derivatised at both its reducingand non-reducing end.

Methods for attaching polysaccharides to proteins are well known in theart and are described in more detail in WO92/22331 and WO-A-0187922. Thepreferred methods in this invention are described in more detail below.Methods are also described in FIGS. 1 and 2 of this Application.

The polysaccharide may be linked to the EPO or EPO-like proteindirectly, i.e. as shown in FIGS. 1 and 2, or via a linker. Suitablelinkers are derived from N-maleimide, vinylsulphone, N-iodoacetamide,orthopyridyl or N-hydroxysuccinimide-containing reagents. The linker mayalso be biostable or biodegradable and comprise, for instance, apolypeptide or a synthetic oligomer. The linker may be derived from abifunctional moiety, as further described in WO2005/016973. A suitablebifunctional reagent is, for instance, Bis-NHS. The reagent may havegeneral formula Z—R¹—Z wherein each Z is a functional group and may bethe same or different and R¹ is a bifunctional organic radical.Preferably, R¹ is selected from the group consisting of alkanediyl,arylene, alkarylene, heteroarylene and alkylheteroarylene, any of whichmay substituted and/or interrupted by carbonyl, ester, sulfide, ether,amide and/or amine linkages. Particularly preferred is C₃-C₆ alkanediyl.Most preferably, R¹ corresponds to the appropriate portion of thesuitable bifunctional reagent.

A preferred compound of this invention is of general formula (I)

wherein m is at least one;

-   -   XB is derived from B—XH which is EPO or an EPO-like protein        wherein XH is NH₂ or SH;    -   L is a bond, a linking group, or comprises a polypeptide or a        synthetic oligomer;    -   GlyO is an anionic saccharide unit;    -   wherein the linking group, if present, is of general formula        —Y—C(O)—R¹—C(O)—; wherein Y is NR² or NR²—NR² and R¹ is a        bifunctional organic radical as defined above; and R² is H or        C₁₋₆ alkyl.

In this aspect of the invention the EPO is linked to the non-reducingend of the polysaccharide. The terminal polysaccharide unit is a sialicacid unit. The other saccharide units in the polysaccharide arerepresented by GlyO and may be the same or different. Suitablesaccharide units include heparin, hyaluronic acid and chondroitinsulphate.

When the EPO is attached directly to the polysaccharide, the group L isa bond. However, the group L may alternatively be derived from anN-maleimide, vinylsulphone, N-iodoacetamide, orthopyridyl orN-hydroxysuccinimide containing reagent. The reagent may have generalformula Z—R¹—Z as defined above. In this embodiment, L is typically agroup

Preferably, XH is NH₂ and is the N-terminus of the EPO or EPO-likeprotein. Alternatively, NH₂ may be the primary amine of a lysine aminoacid side chain. In a different embodiment, XH is a thiol group, SH, ofthe side chain of a cysteine amino acid.

Another aspect of the invention is a pharmaceutical compositioncomprising a novel compound as defined above and one or morepharmaceutically acceptable excipients.

The pharmaceutical composition may be in the form of an aqueoussuspension. Aqueous suspensions contain the novel compounds in admixturewith excipients suitable for the manufacture of aqueous suspensions. Thepharmaceutical compositions may be in the form of a sterile injectableaqueous or homogeneous suspension. This suspension may be formulatedaccording to the known art using suitable dispersing or wetting agentsand suspending agents.

Pharmaceutical compositions may be administered orally, intravenously,intraperitoneally, intramuscularly, subcutaneously, intranasally,intradermally, topically or intratracheally for human or veterinary use.

The compositions may further comprise a formulation additive. Byformulation additive we mean an excipient which is capable ofstabilising the EPO either internally or externally, as described inWang et al (1999). The excipient may be a stabiliser, a solubilser or ametal ion. Suitable examples of formulation additives include one ormore buffers, stabilisers, surfactants, salts, polymers, metal ions,sugars, polyols or amino acids. These may be used alone or incombination.

Stabilisers typically act by destabilisation of the denatured state of aprotein leading to increased Gibbs free energy change for unfolding ofthe protein. The stabiliser is preferably a sugar or a polyol, forexample sucrose, sorbitol, trehalose, glycerol, mannitol, lactose andethylene glycol. A stabilising buffer is sodium phosphate.

The solubiliser is preferably a surfactant, preferably a non-ionicsurfactant. Suitable examples include Tween 80, Tween 20, Tween 40,Pluoronic F68, Brij 35 and Triton X100.

The metal ion is preferably divalent. Suitable metal ions include Zn²⁺,Ni²⁺, Co²⁺, Sr²⁺, Cu²⁺ and Fe²⁺.

The formulation additive may also be a polymer selected from human serumalbumin, PSA, PEG or hydroxy-beta-cyclodextrin.

Suitable amino acids and amino acid derivatives for use as theformulation additive include histidine, glycine, other similar aminoacids and sodium aspartate.

Another aspect of this invention is a composition comprising apopulation of anionic polysaccharide derivatives of EPO or an EPO-likeprotein, wherein the derivatives comprise between 2 and 125 saccharideunits and wherein the population consists of substantially onlyN-terminal derivatives of the protein. By “population” we mean thatthere is more than one polysaccharide derivative in the composition. Thederivatives may comprise the same or different numbers of saccharideunits. Preferably, the polydispersity of the polysaccharide in thecomposition is less than 1.3, more preferably less than 1.1. Preferredpolysaccharides are as detailed above for the other aspects of thisinvention.

In the population, substantially all of the EPO is derivatised at theN-terminus only. By this, we mean that 85%, preferably at least 90%,most preferably at least 95% of the protein in the population isderivatised with PSA at the N-terminus only.

The degree of derivatisation at the N-terminus may be determined bytechniques known in the art such as peptide mapping or EdmanDegradation.

A further aspect of the invention is a compound as described above foruse in therapy.

In accordance with a final aspect of the invention, we provide a methodfor producing a polysaccharide derivative of EPO or of an EPO-likeprotein wherein an anionic polysaccharide comprising 2-200 saccharideunits is chemically reacted with the EPO or EPO-like protein.

It will be noted in this aspect of the invention, the polysaccharide mayreact at any group on the EPO or EPO-like protein. For instance, thepolysaccharide may react with an amine, hydroxyl, carboxyl or sulfhydrylgroup. Preferably, the group is an amine group, more preferably aterminal amine group. The amine may alternatively be the amine sidechain of an amino acid, such as a lysine amino acid. The polysaccharidemay also react at any carbohydrate residues on the EPO, such as onpendant glycone groups.

Polysaccharides may be linked to amino acid side chains by methods knownon the art. For instance, a polysaccharide may be coupled to theC-terminal, —COOH or carboxyl side chains of Asp or Glu by in vitrocoupling. Thiol groups of cysteine amino acids may also be linked topolysaccharides by in vitro coupling. These methods are describedfurther in WO03/055526, in particular the table on pages 6 and 7. Inthis reference, in vitro coupling is also used to link anoligosaccharide moiety to the amide group on the side chain of Gln. Invitro imidazole groups of Arg and His residues respectively are alsodescribed. Each of these methods may be used to derivatise the EPO ofthe present invention.

The polysaccharide may also react with a modified form of EPO. Forinstance, one or more groups on the EPO may have undergone a chemicaltransformation, for instance, by reduction or oxidation. A reactivecarbonyl may be generated in the place of the terminal amino group ofEPO using oxidation conditions, for instance.

Suitable polysaccharides for use in the method of this invention are asdescribed previously for the novel compounds.

The compounds of the invention may be manufactured by any of thesuitable methods described in the prior art. For example, a typicalmethod is described to our previous Patent Application WO92/22331.

Typically, the anionic polysaccharide has been activated beforederivatisation to EPO. It may, for instance, have a reactive aldehydegroup and the derivatisation reaction may be carried out under reducingconditions. The reactive aldehyde group may be produced by controlledoxidation of a hydroxyl group of the polysaccharide. Most preferablythis reactive aldehyde is generated in a preliminary step, in which thepolysaccharide is reacted under controlled oxidation conditions, forinstance using sodium periodate, in aqueous solution. Preferably theoxidation is a chemical oxidation, although enzymes which are capable ofcarrying out this step may also be used. The reactive aldehyde group maybe at the non-reducing end or reducing end of the polysaccharide. TheEPO, typically the N-terminus, may then react with the reactive aldehydegroup to produce an adduct which, when reduced, produces the N-terminalderivative of EPO.

The activation of the polysaccharide should preferably be carried outunder conditions such that there is substantially no mid-chain cleavageof the backbone of the polysaccharide, that is substantially nomolecular weight reduction. The oxidant is suitably perrhuthenate, or,preferably, periodate. Oxidation may be carried out with periodate at aconcentration in the range 1 mM to 1M, at a pH in the range 3 to 10, atemperature in the range 0 to 60° C. for a time in the range 1 min to 48hours.

Suitable reducing conditions for the derivatisation reaction may utilisehydrogen with catalysts or, preferably hydrides, such as borohydrides.These may be immobilised such as AMBERLITE™ (strong acid, gel-typecation exchange resin)-supported borohydride. Preferably alkali metalhydrides such as sodium borohydride is used as the reducing agent, at aconcentration in the range 1 μM to 0.1M, a pH in the range 5.0 to 10, atemperature in the range 0 to 60° C. and a period in the range 1 min to48 hours. The reaction conditions are selected such that pendantcarboxyl groups on the starting material are not reduced. Other suitablereducing agents are cyanoborohydride under acidic conditions, e.g.polymer supported cyanoborohydride or alkali metal cyanoborohydride,L-ascorbic acid, sodium metabisulphite, L-selectride,triacetoxyborohydride etc.

Other activated derivatives of polysaccharides may have utility in thepresent invention, including those with pendant functional groups suchas NHS, as described in our earlier Patent Application WO06/00540.

In one embodiment, the reactive aldehyde is at the reducing end of thepolysaccharide and the non-reducing end has been passivated such that itdoes not react with pendant groups on the EPO.

The reactivity of the reducing end of colominic acid, though weaktowards protein targets, is sufficient to be troublesome in themanufacture of chemically defined conjugates.

Chemistry suitable for preparing a polysaccharide with a reactivealdehyde at the reducing terminal of a polysaccharide is described inour earlier Application WO05/016974. The process involves a preliminaryselective oxidation step followed by reduction and then furtheroxidation to produce a compound with an aldehyde at the reducingterminal and a passivated non-reducing end.

WO2005/016973 describes polysialic acid derivatives that are useful forconjugation to proteins, particularly those which have free sulfhydryldrugs. The polysialic acid compound is reacted with a heterobifunctionalreagent to introduce a pendant functional group for site-specificconjugation to sulfhydryl groups. The anionic polysaccharides used inthe present invention may also be derivatised with a heterobifunctionalreagent in this manner.

The polysaccharide may be derivatised before it reacts with EPO. Forinstance, the polysaccharide may react with a bifunctional reagent.

The polysaccharide may be subjected to a preliminary reaction step, inwhich a group selected from a primary amine group, a secondary aminegroup and a hydrazine is formed on the terminal saccharide, which ispreferably sialic acid, followed by a reaction step in which this isreacted with a bifunctional reagent to form a reaction-intermediate, asfurther described in WO2006/016168. The intermediate may then react withthe EPO. The bifunctional reagent may have general formula Z—R¹—Z, asdefined previously.

We have found that certain reaction conditions promote selectivederivatisation at the N-terminal of the EPO. To promote selectivereaction at the N-terminal, the derivatisation reaction should becarried out in a first aqueous solution of acidic pH, and the resultantpolysaccharide derivative should then be purified in a second aqueoussolution of higher pH than the first aqueous solution. Typically the pHof the first aqueous solution is in the range 4.0-6.0 and the pH of thesecond aqueous solution is in the range of 6.5-9.0, preferably 6.5-8.5or 6.5-8.0. The low pH of the derivatisation reaction promotes selectivederivatisation at the N-terminus of the protein rather than at anymid-chain sites.

Furthermore, we have found that the use of certain formulation additivespromotes the formation of a selective, stable, polysaccharideEPO-derivative. The formulation additive may be selected from one ormore buffers, stabilisers, surfactants, salts, polymers, metal ions,sugars, polyols or amino acids. These may be added to the reactionmedium, or alternatively may be added to the final product composition,as a stabiliser.

In one embodiment of this invention, the formulation additive issorbitol, trehalose or sucrose. In a different embodiment, theformulation additive is a non-ionic surfactant. The formulation additivemay alternatively be a polymer selected from PSA, PEG orhydroxy-beta-cyclodextrin. In a different embodiment the formulationadditive is a divalent metal ion. Preferred divalent metal ions includeZn²⁺, Ni²⁺, Co²⁺, sr²⁺, Fe²⁺, Mg²⁺ or Ca²⁺.

The formulation additive may be a buffer. Preferably when theformulation additive is a buffer, it is sodium phosphate.

The purification of the polysaccharide derivative in the method of thepresent invention may be carried out using a variety of methods known inthe art. Examples of suitable purification methods include HIC(hydrophobic interaction chromatography), SEC (size exclusionchromatography), HPLC (high performance liquid chromatography), AEC(anion exchange chromatography) and metal affinity chromatography.

A population of polysialic acids having a wide molecular weightdistribution may be fractionated into fractions with lowerpolydispersities, i.e. into fractions with differing average molecularweights. Fractionation is preferably performed by anion exchangechromatography, using for elution a suitable basic buffer, as describedin our earlier Patent Applications WO2005/016794 and WO2005/03149. Thefractionation method is suitable for a polysaccharide starting materialas well as to the derivatives. The technique may thus be applied beforeor after the essential process steps of this invention. Preferably, theresultant polysaccharide derivative of EPO has a polydispersity of lessthan 1.3, more preferably less than 1.2, most preferably less than 1.1.

The derivatisation of EPO in accordance with this invention, results inincreased half-life, improved stability, reduced immunogenicity, and/orcontrol of solubility of the protein. Hence the bioavailability and thepharmacokinetic properties of EPO are improved. The new method is ofparticular value for creation of a monopolysialylated-EPO conjugate.

The invention is illustrated by Examples 1 to 3.12 and by reference tothe following drawings:—

FIG. 1 is a reaction scheme showing the prior art activation of thenon-reducing sialic acid terminal unit;

FIG. 2 is a reaction scheme showing the N-terminal or randomderivatisation of proteins;

FIG. 3 a shows the degradation of 24 kDa colominic acid (CA) atdifferent pHs using Triple Detection GPC (Viscotek:RI+RALS+Viscosiometer);

FIG. 3 b shows Gel Permeation chromatography of the CA polymer;

FIG. 4 shows the characterisation of PEGylated and polysialylated EPO bySDS-PAGE;

FIGS. 5 a and 5 b show the characterisation of polysialylated EPO bySDS-PAGE and SE-HPLC;

FIGS. 6 a-d show the characterisation of EPO, polysialylated andPEGylated EPO by SE-HPLC;

FIG. 7 shows the FACS data for EPO (reticulocytes count);

FIG. 8 shows in vivo clearance of EPO formulations;

FIG. 9 shows in vivo clearance of EPO formulations;

FIG. 10 shows characterization of EPO-CA conjugates by SE-HPLC;

FIG. 11 shows in vivo clearance of non glycosylated EPO vs.polysialylated non glycosylated EPO;

FIG. 12 shows in vivo clearance of non glycosylated EPO vs.polysialylated non glycosylated EPO;

FIG. 13 shows characterisation of NG-EPO-CA conjugates by SE-HPLC andSDS PAGE;

FIG. 14 shows detection of EPO PSA by sandwich ELISA;

FIG. 15 shows sensitivity of ELISA to EPO-PSA in reagent diluent;

FIG. 16 shows stability of EPO conjugates by size exclusion HPLC;

FIG. 17 shows SDS-PAGE of polysialylated EPO;

FIG. 18 shows in vivo efficacy of EPO formulations (n=3-4±SE) to outbredfemale mice; 12 weeks old; SC (aldehyde chemistry);

FIG. 19 in vivo efficacy of EPO formulations (Female Wistar rats; 8-9weeks old; n=5±SEM); and

FIG. 20 shows PEGylation of NG EPO by SE-HPLC.

EXAMPLES

Materials

Ammonium carbonate, ethylene glycol, polyethylene glycol (8KDa), sodiumcyanoborohydride (>98% pure), sodium meta-periodate and molecular weightmarkers, ammonium sulphate, sodium chloride, sodium phosphate, sorbitol,Tween 20 and Tris were obtained from Sigma Chemical Laboratory, UK.Sodium acetate and sodium phosphate were from BDH, UK. The colominicacid used, linear alpha-(2,8)-linked E. coli K1 polysialic acids (22.7kDa average, high polydispersity 1.34, 39 kDa p.d. 1.4; 11 kDa, p.d.1.27) was from Camida, Ireland and S.I.I.L. India Ltd. Other materialsincluded 2,4 dinitrophenyl hydrazine (Aldrich Chemical Company, UK),dialysis tubing (3.5 KDa and 10 KDa cut off limits; MedicellInternational Limited, UK), Sepharose SP HiTrap, PD-10 columns, Q FF[column 1 ml or 5 ml]; Hitrap Butyl HP column [1 or 5 ml]; (Pharmacia,UK), Tris-glycine polyacrylamide gels (4-20% and 8-16%), Tris-glycinesodium dodecylsulphate running buffer and loading buffer (Novex, UK).Deionised water was obtained from an Elgastat Option 4 waterpurification unit (Elga Limited, UK). All reagents used were ofanalytical grade. A plate reader (Dynex Technologies, UK) was used forspectrophotometric determinations in protein or CA assays. Mice and ratswere purchased from Harlan, UK and acclimatised for at least one weekprior to their use. EPO was obtained from SIIL, India,

1. Protein and Colominic Acid Determination

Quantitative estimation of polysialic acids (as sialic acid) with theresorcinol reagent was carried out by the resorcinol method[Svennerholm, 1957] as described elsewhere [Gregoriadis et al., 1993;Fernandes and Gregoriadis, 1996, 1997]. Protein was measured by the BCAcolorimetric method or UV absorbance at 280 nm.

2.1. Activation of Colominic Acid

Freshly prepared 0.02 M sodium metaperiodate (NaIO₄) solution (8 foldmolar excess) was mixed with CA at 20° C. and the reaction mixture wasstirred magnetically for 15 min in the dark. A two-fold volume ofethylene glycol was then added to the reaction mixture to expend excessNaIO₄ and the mixture left to stir at 20° C. for a further 30 min. Theoxidised colominic acid was dialysed (3.5 KDa molecular weight cut offdialysis tubing) extensively (24 h) against a 0.01% ammonium carbonatebuffer (pH 7.4) at 4° C. Ultrafiltration (over molecular weight cut off3.5 kDa) was used to concentrate the CAO solution from the dialysistubing. Following concentration to required volume, the filterate waslyophilized and stored at −40° C. until further use. Alternatively, CAOwas recovered from the reaction mixture by precipitation (twice) withethanol.

2.2. Determination of the Oxidation State of CA and Derivatives

Qualitative estimation of the degree of colominic acid oxidation wascarried out with 2,4 dinitrophenylhydrazine (2,4-DNPH), which yieldssparingly soluble 2,4 dinitrophenyl-hydrazones on interaction withcarbonyl compounds. Non-oxidised (CA)/oxidised (CAO) were added to the2,4-DNPH reagent (1.0 ml), the solutions were shaken and then allowed tostand at 37° C. until a crystalline precipitate was observed [Shrineret. al., 1980]. The degree (quantitative) of CA oxidation was measuredwith a method [Park and Johnson, 1949] based on the reduction offerricyanide ions in alkaline solution to ferric ferrocyanide (Persianblue), which is then measured at 630 nm. In this instance, glucose wasused as a standard.

2.3. Gel Permeation Chromatography

Colominic acid samples (CA and CAO) were dissolved in NaNO₃ (0.2M),CH₃CN (10%; 5 mg/ml) and were chromatographed on over 2×GMPW_(XL)columns with detection by refractive index (GPC system: VE1121 GPCsolvent pump, VE3580 RI detector and collation with Trisec 3 softwareViscotek Europe Ltd). Samples (5 mg/ml) were filtered over 0.45 μm nylonmembrane and run at 0.7 cm/min with 0.2 M NaNO₃ and CH₃CN (10%) as themobile phase.

The results are shown in FIG. 3 b and tables 5 and 6.

2.4. Colominic Acid Stability

The rules for chemistry of the PEGylation cannot be applied topolysialylation as such because of the difference in the physiochemicalproperties of these molecules. PSA is an acid labile polymer and isstable for weeks around neutral pH (FIG. 3 a). The results in FIG. 3 ashow that at pH 6.0 and 7.4 CA is stable for 8 days, at pH 5.0 there isslow degradation (after 48 hours 92% of initial MW), and at pH 4.0 thereis slow degradation (after 48 hours 70% of initial MW). Polysialic acidis highly hydrophilic whereas PEG is amphiphilic. When thepolysialylation is carried out using conditions used for PEGylation,aggregation and precipitation of the proteins is seen in many cases.

3. Preparation of N-Terminal Protein-CA Conjugates with FormulationAdditives

3.1. Preparation of EPO-CA Conjugates (N-Terminal Method)

EPO was supplied as a solution (0.34 mg/ml in 10 mM sodium phosphatebuffer 130 mM NaCl pH 7.0; specific activity: 110,000 U/mg, m.w. 30600)and stored at −32° C., protein was defrosted at 2-8° C. and requiredamount was taken into a 2 ml eppendorf tube. The required amount (25fold molar excess) of colominic acid was taken and the protein solutionwas added to solid CA and mixed gently. The required volume of sodiumcyanoborohydride solution was added to have 50 mM or 3.17 mg/ml in thereaction mixture, vortex and check the pH of the final reaction mixture;if necessary adjust the pH to 6.0. Tube was sealed and stirred atdesired temperature for 24 hours or the reaction mixture was firstincubated at RT (22° C.) for 8 hours and then kept at 4±1° C. overnight(14 hours). After incubation, the necessary samples were taken (e.g. foractivity assay, SDS-PAGE, SE-HPLC).

3.1.1 Purification and Characterization of EPO-CA Conjugates (N-TerminalMethod)

The remaining reaction mixture sample was diluted with HIC buffer A1 (3M Ammonium sulphate, pH 6.3) so that a final concentration of 2 Mresults and loaded on the HIC column previously equilibrated with HICbuffer A at the rate of 1.5 ml/min at RT. Loading fraction collected andlabeled. Column was washed with HIC buffer A2 (2M Ammonium sulphate, pH6.3) (at least 6 column volume) fractions collected and labeled. Theproduct was eluted with HIC buffer B (50 mM Na₂HPO₄ pH 7.4), firstfraction (0.5 ml) and then 0.5-1 ml fractions (6CV) were collected andlabeled. Samples were kept on ice (4±1° C.) during purification.

The protein concentration was analysed by UV (280 nm) (Abs of 1 mg/ml ofEPO is about 0.743). The samples were taken for SDS-PAGE. The separationof non-conjugated EPO was performed using anion exchange chromatography(AEC) if CA Mw is too small (e.g. 22 KDa) for separation of conjugateand EPO by SE-HPLC. For AEC the HIC fractions containing protein werediluted with AEC buffer A (50 mM Tris, 150 mM NaCl pH 8.0) (1 mlsample+5 ml AEC buffer A) and loaded to the EC column pre-equilibratedwith AEC buffer A at 1.0 ml/min. Loading fractions were collected andlabeled. Column was washed with AEC buffer A (at least 10 ml) fractionscollected and labeled. Eluted the product with elution buffer B (50 mMTris, 600 mM NaCl, pH 8.0), first fraction of 0.5 ml and then 0.5-1 mlfractions at 2.0 ml/min were collected and labeled. Samples were kept onice during purification.

Alternatively purification can be done by SE-HPLC (e.g. to separateconjugates from EPO if CA used has high molecular weight, e.g. 39 kDa).The protein concentration was analysed by UV (280 nm) (Abs of 1 mg/ml ofEPO is about 0.743). Samples were taken for SDS-PAGE.

An aliquot was removed for protein assay and CA assay. The remainingsolution was stored at −20° C. until use. Products were characterised bySDS-PAGE. To determine the activity of EPO and NG EPO samples ininducing proliferation in vitro of erythrocyte progenitor cells isolatedfrom the spleen of a mouse rendered anaemic artificially through I.P.injection of phenylhydrazine was used. The protocol was adapted based onthe method reported by Krystal [1972]. The assay depends on adding EPOto erythrocyte progenitors and measuring the rate of DNA replication bydetermining the rate of incorporation of ³H-thymidine. The in vivopharmacokinetics (PK) and pharmacodynamics (PD) studies were done inB6D2F1 mice.

3.2. Preparation of EPO-CA Conjugates (Random)

EPO was supplied as a solution (0.34 mg/ml in 10 mM sodium phosphatebuffer 130 mM NaCl pH 7.0; specific activity: 110,000 U/mg, m.w. 30600)and stored at −32° C., protein was defrosted at 2-8° C. and requiredamount was taken into a 2 ml Eppendorf tube. The required amount ofcolominic acid was taken and the protein solution was added to solid CAand mixed gently. The required volume of sodium cyanoborohydridesolution was added to have 50 mM or 3.17 mg/ml in the reaction mixture,vortex and check the pH of the final reaction mixture; if necessaryadjust the pH to 7.4. The tube was sealed and stirred at desiredtemperature (4±1° C.) for 24 hours. After incubation, the necessarysamples were taken (e.g. for activity assay, SDS-PAGE, SE-HPLC).

3.2.1. Purification and Characterization of EPO-CA Conjugates (Random)

The remaining reaction mixture sample was diluted with HIC buffer A1 (3M Ammonium sulphate, pH 6.3) so that a final concentration of 2 Mresults and loaded on the HIC column previously equilibrated with HICbuffer A at the rate of 1.5 ml/min at RT. Loading fraction was collectedand labeled. The column was washed with HIC buffer A2 (2 M Ammoniumsulphate, pH 6.3) (at least 6 column volume), washing fractions werecollected and labeled. Eluted the product with HIC buffer B (50 mMNa₂HPO₄ pH 7.4), first fraction of 0.5 ml and then 0.5-1 ml fractions(6CV) were collected and labeled. Samples were kept on ice (4±1° C.)during purification.

The protein concentration was analysed by UV (280 nm) (Abs of 1 mg/ml ofEPO is about 0.743). The samples were taken for SDS-PAGE. The separationof non-conjugated EPO was performed using anion exchange chromatography(AXC) if CA Mw is too small (e.g. 22 KDa) for separation of conjugateand EPO by SE-HPLC. For AXC the HIC fractions containing protein werediluted with AXC buffer A (50 mM Tris, 150 mM NaCl pH 8.0) (1 mlsample+5 ml AXC buffer A) and loaded to the AXC column pre-equilibratedwith AXC buffer A at 1.0 ml/min. The loading fractions were collectedand labeled. Column was washed with AXC buffer A (at least 10 ml)fractions were collected and labeled. The product was eluted withelution buffer (50 mM Tris, 600 mM NaCl, pH 8.0), first fractioncollected (0.5 ml) and then 0.5-1 ml fractions at 2.0 ml/min andlabeled. Samples were kept on ice during purification.

Alternative purification can be done by SE-HPLC (e.g. to separateconjugates from EPO if CA used has high molecular weight, e.g. 39 kDa).The protein concentration was analysed by UV (280 nm) (Abs of 1 mg/ml ofEPO is about 0.743). The samples were taken for SDS-PAGE.

3.3. Glycon Chemistry

Hydrazide colominic acid was dissolved in the EPO solution to get thefinal CA concentration of 10 mM. The pH of the solution was adjusted to5.5. The required volume of NaIO₄ solution in NaOAc solution was addedto get the final concentration of 5 mM NaIO₄. The reaction was stoppedwith NaHSO3 (final concentration of NaHSO3 to be 20 mM). The reactionmixture was incubated at room temperature. Finally the required volumeof NaCNBH3 solution in NaOAc solution was added to give the finalconcentration of 50 mM NaCNBH3. The reaction was continued at 4±1° C. onshaker for one hour. After incubation the necessary samples were takenfor SDS, SE-HPLC, activity assay.

3.3.1. Purification and Characterization of EPO-CA Conjugates (GlyconChemistry)

The remaining reaction mixture sample was diluted with HIC buffer A1 (3M Ammonium sulphate, pH 6.3) so that a final concentration of 2 Mresults and loaded on the HIC column previously equilibrated with HICbuffer A at the rate of 1.5 ml/min at RT. Loading fraction collected andlabeled (L₁-L_(x)). The column was washed with HIC buffer A2 (2 MAmmonium sulphate, pH 6.3) (at least 6 column volume) and the fractionswere collected and labeled. The product was eluted with HIC buffer B (50mM Na₂HPO₄ pH 7.4), first fraction of 0.5 ml and then 0.5-1 ml fractions(6CV) were collected and labeled. Samples were kept on ice (4±1° C.)during purification.

The protein concentration was analysed by UV (276 nm) (Abs of 1 mg/ml ofEPO was about 0.743). The samples were taken for SDS-PAGE. Theseparation of non-conjugated EPO was performed using anion exchangechromatography (AXC) if CA Mw is too small (e.g. 22 KDa) for separationof conjugate and EPO by SE-HPLC. For AXC the HIC fractions containingprotein were diluted with AXC buffer A (50 mM Tris, 150 mM NaCl pH 8.0)(1 ml sample+5 ml AXC buffer A) and loaded to the AXC columnpre-equilibrated with AXC buffer A at 1.0 ml/min. The loading fractionswere collected and labeled. The column was washed with AXC buffer A (atleast 10 ml) and the fractions were collected and labeled. The productwas eluted with elution buffer (50 mM Tris, 600 mM NaCl, pH 8.0), firstfraction of 0.5 ml and then 0.5-1 ml fractions at 2.0 ml/min werecollected and labeled. Samples were kept on ice during purification.

Alternative purification can be done by SE-HPLC (e.g. to separateconjugates from EPO if CA used has high molecular weight, e.g. 39 kDa).The protein concentration was analysed by UV (280 nm) (Abs of 1 mg/ml ofEPO is about 0.743). The samples were taken for SDS-PAGE.

3.4. PEGylation of EPO:

EPO (30.6 kDa) was supplied as a solution (0.954 mg/ml in 10 mM sodiumacetate buffer, pH 4.0 containing 5% sorbitol, 0.025 mg/ml polysorbate80) and stored at 2-8° C. EPO solution was concentrated to make about1.0 mg/ml of solution. The required amount of EPO was taken into anEppendorf tube and placed on ice. The amount of PEG added forconjugation was calculated based on formula:

${{Weight}\mspace{14mu}{of}\mspace{14mu}{PEG}} = {\frac{{Amount}\mspace{14mu}{of}\mspace{14mu}{protein}\mspace{14mu}(g)}{\left( {{MW}\mspace{14mu}{of}\mspace{14mu}{protein}} \right)} \times \left( {{MW}\mspace{14mu}{of}\mspace{14mu}{PEG}} \right) \times \left( {{Molar}\mspace{14mu}{excess}\mspace{14mu}{of}\mspace{14mu}{PEG}} \right)}$

The required amount of PEG 20K was weighed out. It was solubilised in 10mM NaOAc, 5% sorbitol, pH 5.5 (20% volume of the final reaction volumeas used here), the mixture was gently vortexed until all the PEG haddissolved and then either filtered into a new eppendorf or centrifugedat 4000 rpm for 5 min and the supernatant was transferred to a neweppendorf to remove any aggregated/precipitated material. Requiredamount of EPO protein solution was added to the PEG solution to give a25 fold molar excess of PEG and was gently mixed by keeping the reactionmixture on a gentle shaker at 4±1° C. Required volume of 100 mg/mlNaCNBH3 solution was added in order to have 50 mM or 3.17 mg/ml in thefinal reaction mixture, gently mixed and the pH of the final reactionmixture was checked, and if necessary adjusted to 5.5 with 1 M NaOH/HCLat 4±1° C. Finally the volume of the reaction was adjusted using 20 mMNaOAC, 5% sorbitol, and pH 5.5 to give a protein concentration of 1mg/ml in the reaction mixture. The tube was sealed and stirred atdesired temperature (4±1° C.) for 24 hours. The reaction was stopped byan appropriate method and samples were taken out for in vitro activity,SDS-PAGE (using 4-20% Tris-glycine gel), SE-HPLC (superose 6 column) andchecked the pH of reaction mixture. To eliminate any precipitate thereaction mixture was centrifuged at 13000 rpm for 5 min before SE-HPLCanalysis and purification, preferred buffer for SE-HPLC was 0.1 M sodiumphosphate (pH 6.9). The results are shown in FIG. 5.

3.5 Preparation of N-Terminal Non-Glycosylated Erythropoietin (NGEPO-CA) Conjugates

NG EPO was supplied as a solution (0.18 mg/ml in 20 mM sodium phosphatebuffer 300 mM NaCl pH 6.65; specific activity 100000 U/mg; m.w. 19000)and stored at −32° C., protein defrosted at 2-8° C. and taken therequired amount into a 2 ml eppendorf. The amount of colominic acid(e.g. oxidised or non-oxidised colominic acid) required for conjugationwas calculated. The required amount of colominic acid was weighed outProtein solution was added to solid CA and mixed gently. The requiredvolume of sodium cyanoborohydride solution was added to the reactionmixture so that the final concentration of sodium cyanoborohydrideshould be 50 mM or 3.17 mg/ml in the reaction mixture. The finalreaction mixture was vortexed and checked the pH; if necessary the pHwas adjusted to 7.4. The tube was sealed and stirred at desiredtemperature (4±1° C.) for 24 hours. After incubation, the necessarysamples were taken for activity assay, SDS-PAGE, SE-HPLC etc.

3.5.1 Purification and Characterization of NG EPO-CA Conjugates

The remaining reaction mixture sample was diluted with HIC buffer A (1.2M Ammonium sulphate, pH 6.3) (1 ml sample+4 ml of buffer A) and loadedon the HIC column previously equilibrated with HIC buffer A. The loadingfractions were collected and labeled. The column was washed with HICbuffer A (at least 10 ml). The washing fractions were collected andlabeled. The product was eluted with HIC buffer B, first fraction of 0.5ml and then 0.5-1 ml fractions were collected and labeled. Samples werekept on ice during purification. The protein concentration was analysedby UV (280 nm) (Abs of 1 mg/ml of nEPO was about 0.743). The sampleswere taken for SDS-PAGE. The reaction conditions led to no significantfree NG EPO in the reaction mixture so no further purification wasnecessary. If NG EPO was present in the reaction mixture, the HICfractions containing protein were concentrated using Vivaspin 6 (5000MWCO) and purification was done by SE-HPLC. The protein concentrationwas analysed by UV (280 nm) (Abs of 1 mg/ml of NG EPO is about 0.743).The samples were taken for SDS-PAGE.

An aliquot was removed for protein assay and CA assay. Stored theremainder at −20° C. until use. Product was characterised by SDS-PAGE.

3.6. SE-HPLC of EPO Formulations

HPLC was performed on a Liquid Chromatograph (Jasco) equipped with aJasco, AS-2057 plus autosampler refrigerated at 4° C., and a JascoUV-975 UV/VIS detector. Data was recorded by EZchrom Elite software onan IBM/PC. The SEC samples were analysed with an isocratic mobile phaseof 0.1 M Na phosphate, pH 6.9; on a Superose 6 column (FIG. 5). FIG. 6shows just one peak at RT=76.408, which is attributed to EPO.

The peak table for the SEC shown on the left hand side of FIG. 5 is asfollows:

TABLE 1 Peak RT % Area Species 1 33.896 13.9 Aggregate 2 60.871 85.7CA38K-EPO 3 76.229 0.4 EPO3.7. SDS Polyacrylamide Gel Electrophoresis, Western Blotting & ELISA

SDS-PAGE was performed using 4-20% trisglycine gels. Samples werediluted with either reducing or non reducing buffer and 5.0 ug ofprotein was loaded into each well. The gels were run on a triglycerinebuffer system and was stained with Coomassie Blue. Western blotting wasperformed using anti PSA antibody (FIG. 4). FIG. 4 shows the SDS-PAGE ofEPO formulations (site-specific; N-terminal).

3.8. In Vitro Activity

To determine the activity of EPO samples in inducing proliferation invitro of erythrocyte progenitor cells isolated from the spleen of amouse rendered anaemic artificially through I.P. injection ofphenylhydrazine was used. The protocol was adapted based on the methodreported by Krystal[1972]. The assay depends on adding EPO toerythrocyte progenitors and measuring the rate of DNA replication bydetermining the rate of incorporation of ³H-thymidine.

3.9. Stability Studies

Sterile EPO conjugates were stored in 20 mM sodium phosphate, pH 7.4; 5%sorbitol and 0.025 mg/ml Tween 20; at 4° C. for six weeks. SE-HPLC ofthe samples was performed using SEC columns under following conditions:Injection volume 100 ul, flow rate 0.250 ml/min, running buffer 0.1 Msodium phosphate, pH 6.9.

3.10. In Vivo Efficacy of EPO Formulations

The in vivo efficacy of EPO formulations was studied in female miceB6D2F1, 7-8 weeks old, 5-15 ugs of protein dose (same activity) wasinjected in mice subcutaneously. Animals were divided into seven groupsof four. EPO formulations were given to each animal of each group in thefollowing manner; EPO, EPO-PSA conjugates, PBS, Aranesp (5 μg). 50 μl ofblood was taken from each animal and was analysed by FACS after stainingwith retic count dye (FIGS. 8 and 9).

5 μl of well mixed whole blood was mixed with 1 ml of Retic-Countreagent and incubated at room temperature for 30 minutes in the dark.Samples were then analysed with the help of FACS machine by counting thereticulocytes.

3.11. Elisa

EPO-PSA was captured by the anti-PSA antibody coated over the plate.Captured EPO-PSA was detected with anti-EPO antibody so that only EPOconjugated with PSA was detected.

3.12. In Vivo Clearance

In vivo clearance of EPO formulation was studied on mice. Appropriateamount of protein dose was injected to the mice subcutaneously andintravenously. EPO formulations were radiolabelled with ¹²⁵I and theradioactivity of the blood sample was measured at frequent intervals.

RESULTS Activation of CA and Determination of Degree of Oxidation

Colominic acid (CA), a linear alpha-2,8-linked homopolymer ofN-acetylneuraminic acid (Neu5Ac) residues was used. Exposure ofcolominic acids to oxidation was carried out for 15 min using 20 mMperiodate at room temperature. The integrity of the internal alpha-2,8linked Neu5Ac residues post periodate treatment was analysed by gelpermeation chromatography and the chromatographs obtained for theoxidised (CAO), material was compared with that of native CA. It wasfound that oxidized and native CA exhibit almost identical elutionprofiles, with no evidence that the successive oxidation step give riseto significant fragmentation of the polymer chain.

Quantitative measurement of the oxidation state of CA was performed byferricyanide ion reduction in alkaline solution to ferrocyanide(Prussian Blue) [Park and Johnson, 1949] using glucose as a standard.Table 1 shows that the oxidized colominic acid was found to have agreater than stoichiometric (>100%) amount of reducing agent, i.e. 112mol % of apparent aldehyde content comprising the combined reducingpower of the reducing end hemiketal and the introduced aldehyde (at theother end, non reducing end).

TABLE 2 Degree of oxidation of various colominic acid intermediates inthe double oxidation reaction scheme using glucose as a standard (100%,1 mole of aldehyde per mole of glucose; n = 3 ± s.d). CA species Degreeof oxidation colominic acid (CA)  16.1 ± 0.63 colominic acid-oxidised(CAO) 112.03 ± 4.97 colominic acid-reduced (CAOR) 0; Not detectablecolominic acid-oxidised-reduced-oxidised (CAORO)  95.47 ± 7.11Preparation, Purification and Characterisation of EPO Conjugates

The procedure to prepare and purify colominic acid (CA) conjugates ofErythropoietin (EPO) in an N-terminally selective manner by conductingthe reaction at a reduced pH (pH 5.5) and random pH (7.4) and at 4±1° C.is detailed above. This involves conjugation in the presence of sodiumcyanoborohydride, followed by purification using ion-exchangechromatography (AEX) to remove free EPO followed by removal of CA byhydrophobic interaction chromatography (HIC). The low pH was used tofavour selective derivatisation of the alpha amino group of theN-terminus, and also in order to minimise aggregation of EPO during thereaction. The composition of the final reaction buffer was 5% sorbitol,0.5 mg/ml Tween 20 in 10 mM NaOAc at pH 5.5.

Formation of the EPO-CA conjugates was confirmed by the SE-HPLC (changeof retention time of EPO-CA as compared to EPO; also co-elution of bothmoieties); ion exchange chromatography (binding of conjugates on to theAEC column) and polyacrylamide gel electrophoresis (SDS-PAGE; broadeningand shifting of bands upwards with high m.w. species) (FIG. 4). Thepolysialyted samples were active in vitro and showed vastly superiorprofile (PK and PD) to plain EPO. FIGS. 11 and 12 show in vivo results.

FIG. 5, left hand side, shows the SE-HPLC of EPO-CA 39 kDa conjugationafter 24 hours. Table 3 is the peak analysis table. Characterisationconditions: column Superdex 200, buffer ammonium bicarbonate 0.15 M, pH7.8.

TABLE 3 Peak RT % Ar Species 1 31.421 3.38 Aggregate 2 48.346 80.76CA39-EPO 3 59.204 15.86 EPO

Degree of derivatisation was found to be more in the PEGylated EPO thanthe polysialylated EPO (FIG. 6). This may be due to the inert nature ofPEG and charged nature of sialic acid. Reticulocyte count from FACS datawas more for PSA-EPO conjugate than the EPO (FIG. 7). After purificationof the EPO-CAO conjugate no significant EPO was seen on the chromatogramfrom SEC HPLC and the derivatisation was proved by the change ofretention time on SE-HPLC and broadening and shifting of bands withhigher molecular weight on SDS PAGE. Polysialylation of EPO was alsoshown by western blotting using murine anti PSA antibody. In vivoclearance profile of PSA-EPO conjugate was found to be superior ascompared to EPO (FIG. 8) when given IV and the area under the curve wasincreased by 7.1 fold. Similarly subcutaneous dose of EPO-PSA also showsgreater retention as compared to EPO and the area under the curve wasincreased by 2.5 fold. Polysialylation of EPO was also found to beproportional to incubation time for reaction mixture, molar excess andpH. In vivo clearance profile (intravenous and subcutaneous) forpolysialylated NG EPO was found to be better than the NG EPO (FIGS. 11and 12). In some SDS gels dipolysialylation of EPO was also seen.EPO-PSA conjugates were also confirmed by the ELISA method (FIGS. 14 and15). Erythropoiesis phenomenon was found to be greater with the EPO-PSAconjugate as compared to the EPO and was found to be proportional to themolecular weight of the polymer from 6 to 15 KDa (FIG. 18). 15 KDa wasfound to be the optimal chain length for EPO as with heavier chains ofsialic acid phenomenon of erythropoiesis reduces. This may be due to thenegatively charged nature of the polymer resulting in the repulsion fromthe receptor. This study was confirmed with the help of FIG. 18. Emax ofAranesp was found to be much greater than EPO-PSA which is notclinically good and leads to thrombosis and could cause exhaustion ofbone marrow and was also found to lower the reticulocytes below thebaseline after the treatment. EPO-PSA was found to be vastly superiorthan the EPO and EPO-PSA was found to be as good as EPO-PEG and EPO-PSAalso leads to constant erythropoiesis.

The PSA conjugates were found to be active in the in vitro activityassay. In vivo efficacy study shows that PSA-EPO conjugates are as goodas PEG conjugates and vastly superior to EPO (FIGS. 8 and 9)

Formation of the NG EPO-CA conjugates was confirmed by the SE-HPLC(change of retention time of NG EPO-CA as compared to NGEPO; alsoco-elution of both moieties); ion exchange chromatography (binding ofconjugates on to the AEC column) and polyacrylamide gel electrophoresis(SDS-PAGE; shifting of bands with high m.w. species). The Figures showthat EPO-CA 39 kDa conjugation after 24 hours. The polysialyted sampleswere active in vitro and showed vastly superior profile (PK and PD) toplain NGepo.

FIG. 10 shows the SE-HPLC results. The peak analysis is shown in table 4below. Characterisation conditions—column Superdex 200, buffer ammoniumbicarbonate 0.15 M, pH 7.8.

TABLE 4 Peak RT % Ar Species 1 31.863 3.41 aggregate 2 33.212 6.65aggregate 3 42.667 14.72 (CA)2-nEPO 4 48.571 74.49 CA-nEPO 5 68.183 0.73nEPO

FIG. 12 shows the in vivo clearance results. PSA-NG EPO showed a vastlysuperior profile as compared to NG EPO.

Table 5 shows values of various parameters used and table 6 gives themolecular weight and polydispersity of CA fractions.

TABLE 5 Parameters Values Mn (Da) 26,666 Mw (Da) 27,956 Mz (Da) 31,129Mp (Da) 22,969 Mw/Mn     1.048 IV (dl/g)      0.2395 Rh (nm)     4.683Branches     0.00 Sample Conc (mg/ml)     5.600 Sample Recovery (%)   90.71 dn/dc (ml/g)     0.156 dA/dc (ml/g)     0.000 Mark-Houwink a    0.048 Mark-Houwink logK     0.425

TABLE 6 CA fraction Mw (kDa) pd 475 97.2 1.285 450 52.3 1.109 425 37.91.062 400 28.0 1.048 375 19.0 1.080 *350 14.5 — *300 10.0 — *250 7.0 —

REFERENCES

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The invention claimed is:
 1. A method for producing a polysaccharidederivative of erythropoietin (EPO), comprising: reacting thepolysaccharide with an amine group of an internal amino acid side chainof EPO, wherein the polysaccharide is anionic and comprises between 2and 125 saccharide units and wherein the EPO is derivatised by thepolysaccharide at the reducing terminal unit of the polysaccharide. 2.The method according to claim 1 wherein the anionic polysaccharide has areactive aldehyde group which reacts with the EPO and the derivatisationreaction is carried out under reducing conditions.
 3. The methodaccording to claim 2 wherein the reactive aldehyde is at the reducingend of the polysaccharide and the non-reducing end has been passivatedsuch that it does not react with the EPO.
 4. A method for producing thecompound of formula (I):

wherein m is at least one; XB is B—XH wherein B is EPO and XH is NH₂ andis an amine group of a lysine amino acid side chain of EPO; L is a bondor a linking group; GlyO is an anionic saccharide unit, wherein thelinking group, if present, is of the formula—Y—C(O)—R¹—C(O)—; wherein R¹ is a difunctional organic radical selectedfrom the group consisting of alkanediyl, arylene, alkarylene,heteroarylene and alkylheteroarylene, any of which is optionallysubstituted and/or interrupted by carbonyl, ester, sulfide, ether, amideand/or amine linkages; and wherein Y is NR² or NR²—NR² and R² is H orC₁₋₆ alkyl comprising: reacting the polysaccharide with an amine groupof an internal amino acid side chain of EPO.
 5. The method according toclaim 4 wherein L is a bond or is


6. The method according to claim 4 wherein the EPO is glycosylated andcomprises 2-100 saccharide units.
 7. The method according to claim 4wherein the EPO is non-glycosylated and comprises 80-125 saccharideunits.
 8. The method according to claim 4, wherein the EPO isglycosylated and comprises 10-80 saccharide units.
 9. The methodaccording to claim 4, wherein the EPO is glycosylated and comprises20-60 saccharide units.
 10. The method according to claim 4, wherein theEPO is glycosylated and comprises 40-50 saccharide units.